Enzyme inhibitors, their synthesis, and methods for use

ABSTRACT

Novel compounds are provided that are effective to inhibit the activity of DHUDase or UrdPase. Such compounds have the general formulawhere X is S or Se; Y is &lt;INS-S DATE=&#34;20020402&#34; ID=&#34;INS-S-00001&#34;/&gt;H, &lt;INS-E ID=&#34;INS-S-00001&#34;/&gt;I, F, Cl, Br, methoxy, benzyl, selenenylphenyl, or thiophenyl, and R1 is &lt;INS-S DATE=&#34;20020402&#34; ID=&#34;INS-S-00002&#34;/&gt;H or &lt;INS-E ID=&#34;INS-S-00002&#34;/&gt;an acyclo tail having the general formulawhere R2 is H, CH2 OH or CH2 NH2; R3 is OH, NH2, or OCOCH2CH2CO2H; and R4 is O, S, or CH2.The compounds can be used in pharmaceutical compositions, along with various chemotherapeutic agents to increase the efficacy of the treatment. These compounds can also be used in methods of treating patients by coadministering or sequentially administering the enzyme inhibiting compounds with a chemotherapeutic agent effective to treat cancers, or viral, fungal, bacterial, or parasitic infections. The compounds have further utility in enhancing imaging. Further, they can be administered alone to prevent and/or treat disorders of pyrimidine catabolism and other physiological disorders.

BACKGROUND OF THE INVENTION

The invention relates to novel enzyme inhibiting compounds, theirsynthesis, and their use in treating pathological and physiologicalconditions.

Pyrimidine analogs and pyrimidine nucleosides are widely used aschemotherapeutic agents for cancer and for viral, fungal, bacterial andparasitic infections. Most pyrimidine analogs used in cancerchemotherapy must be convened to the nucleoside 5′-monophosphate levelbefore any anticancer activity can be realized. However, all most allare administered as nucleosides or bases to facilitate transport intocells. The administered compounds are subject to catabolism andinactivation by various enzymes within a patient's body. Pyrimidines,for example, are degraded by the enzymes uridine phosphorylase (UrdPase)and dihydrouracil dehydrogenase (DHUDase). As a result, the balancebetween the anabolic (activation) and catabolic (inactivation) pathwaysmust be considered when designing or choosing a chemotherapeutic regimefor treating various malignancies, or for treating viral, fungal,bacterial or parasitic infections.

Until recently, most studies of pyrimidine analog metabolism havefocused on anabolism, with little attention devoted to catabolism.Pyrimidine bases and nucleoside analogs can be anabolized within apatient's body to the nucleoside 5′-monophosphate, or catabolized toβ-amino acids. The catabolism of nucleosides to bases proceeds bynucleoside phosphonilases. The resulting bases are then convened totheir respective β-amino acids by a chain of three reactions, catalyzedby DHUDase, dihydropyrimidinase and β-ureidopropionase. Wastemack,Pharmac. Ther., 8:629-651 (1981); Naguib, et al, Cancer Res.,45:5405-5412 (1985). Cytidine, cytosine and their analogs must bedeaminated before they can be catabolized.

The importance of DHUDase as a target Ibr chemotherapy has beenestablished by several recent studies. For example, patients receivingcontinuous infusion of 5-fluorouracil (5-FUra) at a constant rate werefound to have plasma concentrations of 5-FUra that varied significantlyduring treatment. This variation followed a circadian rhythm which wasthe inverse of that observed for DHUDase activity. Harris et al,Biochem. Pharmac., 37: 759-4762 (1988); Harris et al, Cancer Res.,49:6610-6614 (1989); Petit E., et al Cancer Res., 48:1676-1679 (1988);Naguib et al, Biochem. Pharmac., 45: 667-673. (1993). That is, highplasma concentration of 5-FUra was associated with low DHUDase activityand vice versa. A significant correlation between the circadian rhythmof DHUDase activity and that of the anticancer efficacy of 5-FUra and5-fluoro-2′-deoxyuridine (5-FdUrd) has also been reported. Petit et al.Cancer Res., 48:1676-1679 (1988); von Roemeling et al, Advances inChronobiology, Part B, 357-373 (1987). Thus it is clear that a strongassociation exists between the level of DHUDase activity and thebioavailability and efficacy of fluoropyrimidines for chemotherapy.

The importance of DHUDase in cancer chemotherapy is further emphasizedby studies with inhibitors of DHUDase, where the inhibitors were foundto increase the concentration and half life of 5-FUra in plasma and todramatically enhance the chemotherapeutic efficacy of 5-FUra in vitroand in vivo. Nevertheless, coadministration of known inhibitors ofDHUDase with 5-FUra has not been popular due to several drawbacksassociated with such previously known inhibitors. Although the knowninhibits enhanced the antitumor activity of 5-FUra, they also served asalternate substrates and caused substantial host-toxicity. Cooper et al,Cancer Res., 32:390-397 (1972); Gentry et al, Cancer Res., 31:909-912(1971). It was also believed that DHUDase inhibition would mimic thegenetic deficiency of this enzyme which is known to be accompanied byneurological disorders. Bakkeren et al, Clinica Chimica Acta,140:246-247 (1984); Tuchman et al N. Engl. J. Med, 313:245-249 (1985);Diasio et al, J. Clin. Invest., 81:47-51 (1988); Wadman et al, Adv. Exp.Med. Biol., 165A: 109-114 (1984). Finally, it was generally believedthat tumors lack or possess very little DHUDase activity. Chaudhury etal, Cancer Res., 18:318-328 (1958); Heidelberger et al, Cancer, Res.,30:1549-1569 (1970); Mukherjee et al, Biol. Chem., 235:433-437 (1960).

Thus, despite the potential promise of DHUDase inhibitors forchemotherapy regimes, currently known inhibitors have demonstratedseveral drawbacks that have discouraged their use in such treatments.

UrdPase inhibitors are also known to possess a number of clinicallyuseful attributes. For example, UrdPase Inhibitors have been proposed toincrease the selectivity and efficacy of various uracil and uridinederivatives in cancer chemotherapy. U.S. Pat. No. 5,077,280 (Sommadossiet al) discloses that UrdPase inhibitors can be used as rescue agents toreduce the toxicity of antiviral agents such as3′-azido-3′-deoxythymidine (AZT), Ideal UrdPase inhibitors are thosethat are potent, specific, and non toxic. Moreover, useful UrdPaseinhibitors should be readily soluble in aqueous solutions bufferedwithin the physiological pH range.

As noted above halogenated pyrimidine bases such as 5-FUra andhalogenated pyrimidine nucleosides such as 5-FdUrd have been used aschemotherapeutic agents in cancer treatments. Because these compoundsare subject to rapid degradation, efficacy of the compound is reduced.Also, the catabolites of these chemotherapeutic agents (e.g.,2-fluoro-β-alanine) are believed to be more toxic to a patient's healthycells.

Halogenated pyrimidine nucleosides, for example, are known to share thesame catabolic pathway as uridine. Because there is little functionalthymidine phosphorylase in many tumor cells, the first step in thecatabolic pathway in tumor cells relies primarily on UrdPase. Theinhibition of this enzyme in tumor cells serves to inhibit thecatabolism of the chemotherapeutic agents in tumor tissue, therebyincreasing their effectiveness. In healthy host tissue, the halogenatedpyrimidine nucleosides can still be catabolized to their pyrimidinecounterparts by the action of thymidine phosphorylase.

Similarly, halogenated pyrimidine bases such as 5-FUra can compete withcellular pyrimidines and their nucleotides for incorporation into RNAand DNA. However, UrdPase inhibitors increase the plasma and uridineconcentration (Monks et al, Biochem. Pharmac., 32, 2003-2009) (1983);Darnowski et al, Cancer Res., 45:5364-5368 (1985)) and the availabilityof uridine for salvage of host healthy tissue. The increase in plasmauridine concentration also increases the pool of uracil nucleosides intissue. The increased intracellular uridine concentration can thusreduce the toxicity of halogenated compounds in host tissue. Moreover,it has been shown that the addition of a phosphorylase inhibitorselectively increases the ability of host tissue to salvage uridine.Darnowski et al, Cancer Res., 45:5364-5368 (1985). This tissue specificenhancement of uridine utilization is of particular importance forchemotherapy regimes using 5-fluorouracil.

Another application of UrdPase inhibitors lies in their use in theprotection against host toxicity of various antiviral agents. Forexample, viral therapies for patients infected with the humanimmunodeficiency virus (HIV) and/or those suffering from Acquired ImmuneDeficiency Syndrome (AIDS) have typically involved the administration ofan antiviral pyrimidine nucleoside such as AZT. Such an antiviral agentfunctions by inhibiting the reverse transcriptase enzyme of the HIV toreduce the cytopathic effects of the virus.

The utility of antiviral pyrimidine nucleosides such as AZT has beenlimited by the toxic effects of AZT or its catabolites such as 3′-amino-3′-deoxythymidine (AMT) on uninfected cells. Cretton et al,Molec., Pharmac., 39:258-266 (1991). Prolonged administration of suchcompounds produces severe side effects including the suppression of bonemarrow growth and severe anemia. The dosage and duration of AZTtherapies is limited because of such complications.

It is now known that uridine and, to some extent, cytidine can reversethe toxic effects of AZT in human bone marrow progenitor cells withoutaffecting the antiviral activity of AZT in infected cells. Sommadossi etal Antimicrob. Agents Chemother., 32, 997-1000 (1988). This rescuingeffect of uridine, although generally beneficial, has disadvantagesbecause of the body's rapid uridine catabolism. Consequently, high dosesare required, and high doses of uridine can cause serious toxic sideeffects, including phlebitis and pyrogenic reactions.

Viral therapies that combine AZT or similar compounds with UrdPaseinhibitors have been suggested in U.S. Pat. No. 5,077,280 (Sommadossi etal). Such treatments utilize UrdPase inhibitors to maintain effectivelevels of uridine in plasma sufficient to rescue uninfected cellswithout requiring the administration of high, potentially harmful dosesof uridine.

Further, a number of synthetic UrdPase inhibitors have been proposed.See Niedzwicki et al, Biochem Pharmac., 31:1857 (1982); Naguib et al,Biochem Pharmac., 36:2195 (1987); Naguib et al, Biochem. Pharmac.,46:1273-1283 (1993). U.S. Pat. No. 4,613,604 (Chu et al); and U.S. Pat.No. 5,141,943 (Naguib et al). Such UrdPase inhibitors include a varietyof substituted acyclouridines and 5-benzyl barbiturate derivatives.

Substituted acyclouridines are good inhibitors of UrdPase, but tend tohave limited water solubility and are difficult and expensive tosynthesize. Water solubility is essential for practical chemotherapy andtreatment of infection in order to enable intravenous administration atphysiological pH ranges and to allow formulation of reasonable volumesto be administered. Unfortunately, some acyclouridines, such as benzylacyclouridine and its derivatives, are soluble only to about 1 mM inwater at room temperature. Administration of a physiologically usefuldose would require dilution of these compounds into excessively largevolumes. 5-Benzyl barbiturate derivatives are also useful UrdPaseinhibitors and have been found to be more water soluble and moredesirable than derivatives of benzyl acyclouridine.

The maintenance of or increase in plasma uridine levels is also usefulto treat several pathological and physiological conditions. For example,uridine has been shown to increase myocardial performance, glucoseuptake, glycogen synthesis and the breakdown of ATP in heart tissue ofrabbits. Plasma uridine level fluctuations also have importantimplications in muscle performance and in myocardial contractility.Further, uridine levels are important in central nervous systemfunctioning. For example, the control of intracellular and plasmauridine levels is believed to have important implications in thetreatment of CNS disorders, including cerebrovascular disorder andconvulsions, epilepsy, Parkinson's and Alzheimer diseases, and seniledementias. Uridine is also potentially useful in the treatment of liverdamage and hepatitis. (See Naguib et al, Biochem. Pharmac. 46:1273(1993) and references cited therein).

It is thus apparent that it is desirable to inhibit the enzymes thatrapidly degrade certain chemotherapeutic agents or that otherwisecontribute to excess uracil or uridine catabolism. In particular,inhibitors of DHUDase and UrdPase are of great relevance to treatmentregimes for cancers as well as viral, fungal, bacterial and parasiticinfections. Further, the control of and maintenance of plasma uridinelevels is thus important in treating and preventing many diseases andpathological conditions. UrdPase Inhibitors can also be used to increaseavailable plasma uridine concentrations. As a result, there is a needfor new and improved enzyme inhibiting compounds, particularlyinhibitors of DHUDase and UrdPase.

Accordingly, it is an object of the invention to provide new compoundsuseful as DHUDase and UrdPase inhibitors. A further object of theinvention is to provide such DHUDase and UrdPase inhibitors which can beused with various chemotherapy regimes to reduce the toxicity ofchemotherapeutic agents to normal and uninfected cells. Another objectof the invention is to provide methods for increasing the efficacy ofchemotherapy regimes in treating cancers as well as viral, fungal,bacterial, and parasitic infections. A further object of the inventionis to increase the efficacy of certain chemotherapeutic regimes whilereducing adverse patient affects associated with such treatments. Yetanother object of the invention is to provide methods for synthesizingsuch new inhibitors of DHUDase and UrdPase. It is also an object of theinvention to provide methods and compositions useful to increase plasmauridine concentrations and effective useful to treat variousphysiological and pathological conditions. A further object of theinvention is to provide methods to treat and/or prevent symptoms ofinherited disorders of pyrimidine catabolism. These and other objects ofthe invention will be apparent from the description that follows.

SUMMARY OF THE INVENTION

The invention relates to novel compounds that are effective asinhibitors of DHUDase or UrdPase. The novel compounds are represented bythe formula

where X is S or Se, Y is H, I, F, Cl, Br, methoxy, benzyl,selenenylphenyl, or thiophenyl; and R₁ is H or an a cyclo acyclo tailhaving the general formula

where R₂ is H, CH₂OH or CH₂NH₂; R₃ is OH, NH₂ or OCOCH₂CH₂CO₂H; and R₄is O, S, or CH₂.

Novel compounds of the invention that inhibit DHUDase include5-(phenylselenenyl)uracil (PSU); 5-(phenylthio)uracil (PTU);5-(phenylselenenyl)barbituric acid; and 5-(phenylthio)barbituric acid.

Preferred compounds of the invention that inhibit UrdPase includecompounds of the above general formulas having an a cyclo tail. Suchcompounds include 1-[(2-hydroxyethoxy)methyl]-5-(phenylselenenyl)uracil(PSAU); 1-[(2-hydroxyethoxy)methyl]-5-(phenylthio)uracil (PTAU);1-[(2-hydroxyethoxy)methyl]-5-(phenylselenenyl)barbituric acid; and1-[(2-hydroxyethoxy)methyl]-5-(phenylthio)barbituric acid.

In another embodiment the invention relates to pharmaceuticalcompositions comprising a chemotherapeutic agent, such as a pyrimidine,in an amount effective to treat cancer or a viral, fungal, bacterial, orparasitic infection; an effective amount of a novel DHUDase or UrdPaseinhibitor of the present invention; and a pharmaceutically acceptablecarrier. The chemotherapeutic agent can be one that is commonly used totreat cancer or viral, fungal, bacterial or parasitic infections andwhich is subject to degradation within a patient's body by DHUDase orUrdPase. Examples of such chemotherapeutic agents include pyrimidinecompounds such as 3′-azido-3′-deoxythymidine;3′-fluoro-3′-deoxythymidine; 2′, 3′-dideoxycytidin-2′-ene;3′-deoxythymidin-2′-ene; 3′-azido-2′,3′-dideoxyuridine;2′,3′-dideoxy-5-fluoro-3′-thiacytidine; 2′,3′-dideoxy-3′-thiacytidine;5-fluoro-2′,3′-dideoxycytidine; 5-fluorouracil;5-fluoro-2′-deoxyuridine; and heterodimers thereof and enantiomersthereof. The chemotherapeutic agent can also be a prodrug of pyrimidinenucleobase analogs, including 1,(2-tetrahydrofuryl)-5-fluorouracil(TEGAFUR); 5-fluorocytosine; 5′-deoxy-5-fluorouridine; and1-ethoxymethyl-5-fluorouracil. The chemotherapeutic agent can also be aprodrug sold by Taiho Pharmaceutical Company, Ltd. of Osaka, Japan underthe tradename UFT, which is a combination of1,(2-tetrahydrofuryl)-5-fluorouracil and uracil.

In another embodiment the invention comprises a method for administeringchemotherapeutic agents while protecting and/or rescuing normal oruninfected cells from any toxicity that may result from theadministration of the chemotherapeutic agent. Further, methods areprovided for improving the efficacy of the chemotherapeutic agent. Themethods of the invention comprise administering the chemotherapeuticagent, and coadministering or sequentially administering a DHUDase orUrdPase inhibiting compound of the type disclosed herein. The inhibitionof the activity of DHUDase or UrdPase prevents or slows the degradationof the chemotherapeutic agent by these enzymes. This prevents or slowsthe degradation of the chemotherapeutic agent also results in lowerlevels of potentially toxic catabolites of the chemotherapeutic agent.These methods thus facilitate a higher concentration and/or a longerhalf-life of the chemotherapeutic agent, thus increasing the efficacy ofthe treatment regime. An additional benefit is that any toxic sideeffects of the chemotherapeutic regime are minimized.

The use of enzyme inhibiting compounds of the present invention are alsoeffective to provide increased plasma levels of natural pyrimidines,such as uridine, which can help to protect and/or rescue healthy cellsfrom toxicity induced by chemotherapeutic agents. The administration ofthese compounds to increase plasma levels of natural pyrimidines canalso be effective to treat pathological and physiological disorders thatrespond to the administration of such pyrimidines. Such disordersresponsive to these treatments include CNS disorders, Parkinson'sdisease, Alzheimer's disease, senile dimentia, sleep disorders, muscledysfunction, long disorders, diabetes, cardiac insufficiency andmyocardial infarction, liver disease and liver damage.

In addition to the novel compounds disclosed herein it has also beendiscovered that several known compounds are effective as UrdPaseinhibitors. The UrdPase inhibiting activity of such compounds waspreviously unknown.

The present invention also contemplates the synthesis of novel enzymeinhibiting compounds such as 5-(phenylselenenyl)uracil and5-(phenylthio)uracil.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to novel compounds that are effective toinhibit the activity of the enzymes dihydrouracil dehydrogenase(DHUDase) or uridine phosphorylase (UrdPase). These compounds are usefulin conjunction with chemotherapeutic regimes that involve theadministration of chemotherapeutic agents that are degraded by DHUDaseor UrdPase to treat cancer, or viral, bacterial, fungal or parasiticinfections. By inhibiting the activity of DHUDase or UrdPase, thecompounds of the present invention are effective to slow or prevent thedegradation of the chemotherapeutic agent by DHUDase or UrdPase. Thisresults in an increase in the concentration and half-life of the agentand thus increases the efficacy of the agent. Further, by slowing orpreventing the degradation of the agents, levels of potentially toxiccatabolites of the agent are significantly reduced and toxic sideeffects associated with many chemotherapeutic regimes are reduced. TheDHUDase and UrdPase inhibitors of the invention can also increaseintracellular levels of natural pyrimidines (e.g., uridine, cytidine,uracil, and thymine) and can be useful to treat pathological andphysiological disorders for which administration of pyrimidines andtheir nucleotides is known to be effective.

The novel compounds of the invention are represented by the formula

where X is S or Se, Y is H, I, F, Cl, Br, methoxy, benzyl,selenenylphenyl, or thiophenyl; and R₁ is H or an a cyclo acyclo tailhaving the general formula

where R₂ is H, CH₂OH or CH₂NH₂; R₃ is OH, NH₂, or OCOCH₂CH₂CO₂H; and R₄is O, S, or CH₂.

Examples of preferred compounds having the above general formula thatare effective to inhibit DHUDase include 5-(phenylselenenyl)uracil(PSU); 5-(phenylthio)uracil (PTU); 5-(phenylselenenyl)barbituric acid;and 5-(phenylthio)barbituric acid.

Examples of preferred compounds having the above general formula thatare effective as inhibitors of UrdPase include5-(phenylselenenyl)acyclouridines and 5-(phenylthio)acyclouridines suchas 1-[(2-hydroxyethoxy)methyl]-5-(phenylselenenyl)uracil (PSAU) and1-[(2-hydroxyethoxy)methyl]-5-(phenylthio)uracil (PTAU), respectively.Examples of other preferred UrdPase inhibiting compounds having theabove general formula include(1-[(2-hydroxyethoxy)methyl]-5-(phenylselenenyl)barbituric acid and1-[(2-hydroxyethoxy)methyl]-5-(phenylthio)barbituric acid.

In addition to novel compounds represented by the general formula shownabove other, previously known compounds have been discovered to beeffective to inhibit UrdPase. Such compounds include5-(phenylselenenyl)uridine; 5-(phenylselenenyl)-2′-deoxyuridine;1-[(2-hydroxyethoxy)methyl]-6-(phenylselenenyl)uridine; 5-(phenylthio)uridine; 5-(phenylthio)-2′-deoxyuridine; and1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)uridine.

As noted above, many useful chemotherapeutic agents are rapidlycatabolized by enzymes such as DHUDase and UrdPase. The rapiddegradation of these agents obviously results in lower efficacy oftreatments involving such agents as their half-life is reduced. Also,the rapid degradation of these compounds yields catabolites that in manycases can be toxic to host tissue. The invention recognizes that theeffectiveness of chemotherapy regimes can be enhanced by using the novelcompounds of the invention, and other enzyme inhibiting compounds, incoadministration or sequential administration with variouschemotherapeutic agents. By inhibiting the enzymes DHUDase or UrdPase,the compounds of the invention are effective to make more of thechemotherapeutic agent available to a patient for a longer period oftime and to minimize or prevent the formation of potentially toxiccatabolites.

The enzyme inhibiting compounds of the present invention can be usedwith a wide variety of chemotherapeutic agents that are effective totreat cancer or to treat viral, fungal, bacterial or parasiticinfections. These compounds typically are pyrimidine compounds such aspyrimidine nucleobases, pyrimidine nucleosides, and prodrugs of suchcompounds. One of ordinary skill in the art will readily appreciate thenumerous chemotherapeutic agents, the efficacy of which can be enhancedby the enzyme inhibiting compounds of the invention.

Examples of pyrimidine bases and pyrimidine nucleosides theeffectiveness of which can be enhanced by the enzyme inhibitors of theinvention include 3′-azido-3′-deoxythymidine;3′-fluoro-3′-deoxythymidine; 2′,3′-dideoxycytadin-2′-ene;3′-deoxythymidin-2′-ene; 5-fluorouracil; 3′-azido-2′,3′-dideoxyuridine;2′,3′-dideoxy-5-fluoro-3-thiacytidine; 2′,3′-dideoxy-3-thiacytidine;5-fluoro-2′,3′-dideoxycytidine; 5-fluoro-2′-deoxyuridine; heterodimersthereof; and enantiomers thereof. Other pyrimidine bases and pyrimidinenucleosides that can be enhanced by the enzyme inhibitors of theinvention include prodrugs of pyrimidine nucleobases analogs. Examplesof such prodrugs include 1,(2-tetrahydrofuryl)-5-fluorouracil;5-fluorocytosine; 5′-deoxy-5-fluorouridine; and1-ethoxymethyl-5-fluorouracil. Another suitable prodrug is one sold byTaiho Pharmaceutical Company, Ltd. of Osaka, Japan under the tradenameUFT, which combines 1,(2 -tetrahydrofuryl)-5-fluorouracil and uracil.

It is also noted that the enzyme inhibiting compounds of the inventionare useful with prodrugs such as 5-fluorocytosine that are administeredto a patient and can be deaminated to useful chemotherapeutic agent(e.g., 5-fluorouracil) by bacterial or fungal enzymes available withincells through transplanted bacterial or fungal genes.

DHUDase inhibitors, in particular, are effective to prevent or slow thecatabolism of various pyrimidine nucleobase analogs (e.g., 5-FUra) ordrugs of pyrimidine nucleobase analogs (e.g.,1,(2-tetrahydrofuryl)-5-fluorouracil; 5-fluorocytosine;5′-deoxy-5-fluorouridine; and 1-ethoxymethyl-5-fluorouracil). Suchinhibitors also prevent or minimize toxicity (e.g, cardiotoxicity,neurotoxicity, hepatotoxicity, and cholestasis) resulting from toxiccatabolites of 5-FUra and its prodrugs (e.g., fluoro-β-alanine and itsbile acid conjugates). The DHUDase inhibitors can also be effective toprevent and treat symptoms of inherited disorders of pyrimidine basecatabolism that result from increased production of β-alanine and itsmetabolites. Such disorders include hyper-β-alaninemia,hypercarnosinuria, and β-alaninuria.

UrdPase inhibitors, in particular, are useful to increase plasma uridinelevels to prevent or minimize toxicity of chemotherapeutic agents usedto treat cancer as well as those used to treat viral, fungal, bacterial,and parasitic infections. The increase of plasma uridine levels can beuseful because suitable plasma uridine levels are effective to preventand/or rescue normal or uninfected host cells from toxicity associatedwith the administration of many chemotherapeutic agents such aspyrimidine nucleobases and pyrimidine nucleosides. The UrdPaseinhibitors can be used alone to increase plasma uridine levels and/orplasma levels of other natural pyrimidines. Alternatively, they can beused in combination with uridine, cytidine, prodrugs or uridine orcytidine, prodrugs of uridine or cytidine nucleosides, and nucleosidetransport inhibitors to increase plasma levels of natural pyrimidinessuch as uridine. The UrdPase inhibitors also prevent or slow thedegradation by UrdPase or various anticancer chemotherapeutic agents andchemotherapeutic agents used to treat viral, fungal, bacterial, andparasitic infections. Further, UrdPase inhibitors can prevent or slowthe degradation of radiosensitizing drugs to enhance imagingcapabilities. Examples of such radiosensitizing drugs include5-iodo-2′-deoxyuridine and 5-bromo- 2′-deoxyuridine.

In another aspect of the invention DHUDase and UrdPase inhibitors of thetype noted herein are useful, by themselves, to increase levels ofnatural pyrimidines such as uridine, cytidine, uracil and thymine. Suchtreatments can be effective to treat pathological and physiologicaldisorders where administration of pyrimidines (e.g., cytidine, uridineand their nucleotides) are useful. Such disorders include CNS disorders,Parkinson's disease, Alzheimer's disease, senile dementia, sleepdisorders, muscle dysfunction, lung disorders, diabetes, cardiacinsufficiency and myocardial infarction, liver disease, and liverdamage. Further details concerning the increase of plasma uridine levelsto treat such disorders are included in copending U.S. patentapplication Ser. No. 106,225, filed Aug. 13, 1993, which is herebyincorporated by reference.

U.S. Pat. Nos. 5,077,280 and 5,141,943, both of which are incorporatedby reference herein, describe various uses of other UrdPase inhibitors.The uses for the UrdPase inhibitors described in these patents are alsoapplicable to the UrdPase inhibitors described herein.

The enzyme inhibiting compounds of the present invention have been foundto be more lipophilic than previously known enzyme inhibitors such asacyclouridines and benzyl barbiturates. Consequently, the beneficial,enzyme inhibiting effects of these compounds can be more rapidlydirected to the liver, the primary site of pyrimidine metabolism withinthe body. The lipophilic nature of these compounds also enables them toremain active within a patient's system for a longer period of time.

The preferred dosages of chemotherapeutic agents are known to those ofordinary skill in the art. The preferred dosages will vary dependingupon numerous factors, including the age, weight and health of thepatient, and the disease to be treated. The potency and potentialtoxicity of a chemotherapeutic agent are additional factors thatinfluence the dosage of a particular chemotherapeutic agent. AZT, forexample, is used to treat AIDS. This drug is effective to inhibit viralreplication when administered in amounts ranging from about 10 mg toabout 100 mg per kilogram of body weight per day. Such dosage units areemployed so that a total of from about 0.7 to about 7 grams of thecompound is administered to a subject of about 70 kg of body weight in a24 hour period. For example, one presently accepted protocol foradministration of the pyrimidine nucleoside AZT calls for 200 mg of AZTto be administered three times per day. The preferred therapeuticdosages of other pyrimidine nucleobases and pyrimidine nucleosides areknown to those skilled in the art.

Chemotherapeutic agents of the type noted herein may be coadministeredor sequentially administered with the enzyme inhibiting compounds of theinvention. The preferred dosages of the enzyme inhibiting compounds ofthe invention range from about 5 to 500 mg/kg/day. A preferred dosage isabout 200 mg/kg per day. One of ordinary skill in the art willappreciate that the dosage of chemotherapeutic agent to be administeredto any given patient can be influenced by the efficacy of the enzymeinhibiting compounds administered to the patient.

The dosage regimen of the combination therapies described aboveobviously may be adjusted to provide the optimum therapeutic response.For example, several divided doses may be administered daily or the dosemay be proportionally reduced as indicated by the exigencies of thetherapeutic situation.

A decided practical advantage provided by this invention is that theactive compounds may be administered in any convenient manner, such asby the oral, intravenous, intramuscular, subcutaneous routes, or byregional infusion.

Pharmaceutical compositions may be prepared by combining a desiredchemotherapeutic agent with a desired DHUDase or UrdPase inhibitor ofthe type disclosed herein.

The active compounds disclosed herein may be orally administered, forexample, with an inert diluent or with an assimilable edible carrier.They may also be enclosed in hard or soft shell gelatin capsules, orthey may be compressed into tablets, or incorporated directly into food.For oral therapeutic administration, the active compounds may beincorporated with excipients and used in the form of ingestible tablets,bucal tablets, troches, capsules, elixirs, suspensions, syrups, wafersand the like. The amount of active compounds in such therapeuticallyuseful compositions is such that a suitable dosage will be obtained.

Pharmaceutical compositions in the form of tablets, troches, pills,capsules and the like may also contain the following: a binder, such asgum tragacanth, acacia, cornstarch, or gelatin; excipients, such asdicalcium phosphate; a disintegrating agent, such as corn starch, potatostarch, aliginic acid and the like; a lubricant, such as magnesiumstearate; and a sweetening agent, such as sucrose, lactose orsaccharine; and a flavoring agent, such as peppermint, oil orwintergreen, or cherry flavoring. When the dosage unit form is acapsule, it may contain, in addition to materials of the above type, aliquid carrier. Various other materials may be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules may be coated with shellac, sugar or both. Asyrup or elixir may contain the active compounds, sucrose as asweetening agent, methyl and propylparabens as preservatives, a dye anda flavoring, such as cherry or orange flavor. Of course, any materialused in preparing any dosage unit form should be pharmaceutically pureand substantially non-toxic in the amounts employed. In addition, theactive compounds may be incorporated into sustained-release preparationsand formulations.

The active compounds may also be prepared in the form of pharmaceuticalcompositions to be administered parenterally or intraperitoneally.Solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereof,and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. Suitable injectable pharmaceutical forms must be sterileand must be fluid to the extent that easy syringability exists. Theymust be stable under the conditions or manufacture and storage and mustbe preserved against the contaminating action of microorganisms, such abacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining for example, water, ethanol, glycerol, propylene glycol, andpolyethylene glycol, and vegetable oils. The proper fluidity can bemaintained, for example, by the use of a coating such as a lecithin, bythe maintenance of the required particle size in the case ofdispersions, and by the use of surfactants. Various antibacterial andantifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid,thimerosal) can be used to prevent the action of microorganisms. In manycases, it will be preferable to include isotonic agent, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents that delay absorption, for example, aluminum monostearate madgelatin.

Sterile injectable solutions can be prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions can be prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredadditional ingredients of the type enumerated above. Sterile powdersused to prepare sterile injectable solutions can be prepared byvacuum-drying and freeze-drying techniques.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art.

5-(Phenylselenenyl)uracil (PSU), 5-(phenylthio)uracil (PRU) and theirderivatives can be prepared through a multistep synthesis scheme. Apreferred starting compound is 5-bromouracil which is reacted withexcess POCl₃ to yield 2,4-dichloro-5-bromopyrimidine. When this compoundis treated with sodium benzylate, preferably in toluene, at roomtemperature, it yields 5-bromo-2,4-bis(benzyloxy)pyrimidine. Lithiationof the 5-bromopyrimidine derivative below −80° C. in dry THF with n-BuLi(1.1 equiv.) generated a C-5 lithiated species. The lithiated speciescan be reacted with either diphenyl diselenide (to obtain PSU) or withdephenyl disulfide (to obtain PTU) (2 equiv.), at a temperature ofapproximately −75° C. for about ½ hour. Quenching the reaction mixturewith glacial AcOH, followed by silica gel column chromatography,providing the corresponding 5-heteraphenyl-2,4-bis(benzyloxy)pyrimidinesin 70 to 75 percent yield. The protecting benzyl groups can be readilycleaved by exposure to trimethylsilyl iodide (2.4 equiv.) in dry CH₂Cl₂at room temperature to give the desired 5-(heteraphenyl)uracils, eitherPSU or PTU, in 78 to 80% yield.

5-phenylselenenyl acyclic nucleosides can be conveniently prepared bydirect electrophilic addition of phenylselenenyl chloride to the acyclicnucleosides in dry pyridine at temperatures above room temperature,preferably at about 60° C. Such synthesis methods are described Schinaziet al, J. Med Chem., 29:1293-1295 (1986), the disclosure of which ishereby incorporated by reference. The products of the electrophilicaddition, obtained as white crystalline compounds, are readily purifiedby chromatography.

The synthesis of these compounds and their utility as enzyme inhibitorsis further described below in Examples 1-9.

EXAMPLE 1

(Synthesis of 5-(Phenylselenenyl)-2,4-bis(benzyloxy)pyrimidine)

To a solution of 5-bromo-2,4-bis(benzyloxy)pyrimidine (742 mg, 2 mmol)in dry THF (10 mL) at −80° C. was added dropwise n-BuLi (1.6M, 1.5 mL,2.4 mmol) with stirring under an argon atmosphere. After the mixture wasstirred for 15 min, diphenyl diselenide (1.25 g, 4 mmol) dissolved inTHF (10 mL) was added and the temperature was maintained below −70° C.After 1 h. at that temperature, the reaction mixture was quenched withglacial AcOH (0.5 mL), and the solution was allowed to warm to roomtemperature. The solution was concentrated to dryness in vacuo, and theresidue was purified by silica gel column chromatography using hexane:CH₂CL₂ (6:4) as eluent to yield a white solid which was crystallizedfrom EtOH to give white needles of5-phenylselenenyl-2,4-bis(benzyloxy)pyrimidine (778 mg, 87%); m.p.66°-68° C.; ¹H NMR (CDCl₃) δ5.38 and 5.42 (2 s, 4H, CH₂), 7.23-7.49 (m,15H, 2 pH and SePh), 8.26 (s, 1H, 6-H). Anal. (C₂₄H₂₀N₂O₂Se) C, H, N.

EXAMPLE 2

(Synthesis of 5-Phenylselenenyluracil (PSU))

To a solution of 5-phenylselenenyl-2,4-bis(benzyloxy)pyrimidine (447 mg,1 mmol) in dry CH₂Cl₂ (10 mL) was added trimethylsilyl iodide (520 mg,2.6 mmol) under anhydrous conditions at room temperature. The yellowsolution was stirred for 1 h. The excess trimethylsilyl iodide wasdestroyed and the intermediate trimethylsilyl ethers limned during thereaction were hydrolyzed by addition of MeOH. The precipitate wasfiltered and the solid crystallized from EtOH to give pure PSU (210 mg,78%); m.p. 249°-251° C.; ¹H NMR (DMSO-d₆) δ7.16-7.37 (m, 5H, SePh), 7.93(s, 1H, 6-H), 11.28 and 11.39 (2 s, 2H, 2 NH, D₂O exchangeable). Anal.(C₁₀H₈N₂O₂Se) C, H, N.

EXAMPLE 3

(Synthesis of 5-(Phenylthio)-2,4-bis(benzyloxy)pyrimidine)

Reaction of 5-bromo-2,4-bis(benzyloxy)-pyrimidine (742 mg, 2 mmol)sequentially with n-BuLi (1.6M, 1.5 mL, 2.4 mmol) and diphenyl disulfide(872 mg, 4 mmol) as described in Example 2 yielded the title compound(630 mg, 79%); m.p. 61°-63° C.; ¹H NMR (CDCl₃) δ5.41 and 5.45 (2 s, 4H,CH₂), 7.06-7.48 (m, 15H, 2 pH and SPh), 8.37 (s, 1H, 6-H ). Anal.(C₂₄H₂₀N₂O₂S) C, H, N.

EXAMPLE 4

(Synthesis of 5-(Phenylthio)uracil (PTU))

Reaction of 5-phenylthio)-2,4-bis(benzyloxy)pyrimidine (400 mg, 1 mmol)with trimethylsilyl iodide (520 mg, 2.6 mmol) in CH2Cl2 (15 mL) asdescribed in Example 2 gave 5-phenylthiouracil (160 mg, 72%); m.p.269°-271′ C. (lit.³⁷ m.p. 272° C.); ¹H NMR (DMSO-d₆) a 7.04-7.25 (m, 5H,SPh), 7.86 (s, 1H, 6-H), 11.32 and 11.41 (2s, 2H, 2 NH, D₂Oexchangeable).

EXAMPLE 5

(Synthesis of 6-(Phenylselenenyl)-2,4-bis(benzyloxy)pyrimidine)

Reaction of 6-bromo-2,4-bis-(benzyloxy)pyrimidine (742 mg, 2 mmol)sequentially with n-BuLi (1.6M, 1.5 mL, 2.4 mmol) and diphenyldiselenide (1.25 g, 4 mmol), as described in Example 2, yielded thetitle compound (590 mg, 66%); m.p. 97°-99° C.; ¹NMR (CDCl₃) δ5.28 and5.39 (2 s, 4H, CH₂), 6.00 (s, 1H, 5-H), 7.26-7.74 (m, 15H, 2 pH andSePh). Anal. (C₂₄H₂₀N₂O₂Se) C, H, N.

EXAMPLE 6

(Synthesis of 6-(Phenylselenenyl)uracil)

Reaction of 6-(phenylselenenyl)-2,4-bis(benzyloxy)pyrimidine (447 mg, 1mmol) with trimethylsilyl iodide (520 mg. 2.6 mmol) in CH₂Cl₂ (15 mL),as described in Example 2, gave the desired product (215 mg, 80%); m.p.238°-240° C.; ¹H NMR (DMSO-d₆) δ4.66 (s, 1H, 5-H), 7.43-7.70 (m, 5H,SePh), 11.16 and 11.28 (2 s, 2 H, D₂O exchangeable). Anal. (C₁₀HN₂O₂Si)C, H, N.

EXAMPLE 7

(Synthesis of 6-(Phenyithio)-2,4-bis(benzyloxy)pyrimidine)

Reaction of 6-bromo-2,4-bis(benzyloxy)-pyrimidine (742 mg, 2 mmol)sequentially with n-BuLi (1.6M, 1.5 mL, 2.4 mmol) and diphenyl disulfide(872 mg, 4 mmol), as described in Example 2, yielded the title compound(610 mg, 76%); m.p. 102°-104° C.; ¹H NMR (CDCl₃) δ5.32 and 5.40 (2 s,4H, CH₂), 5.83 (s, 1H, 5-H), 7.28-7.60 (m, 15H, 2 Ph and SPh) Anal.(C₂₄H₂₀N₂O₂S) C, H, N.

EXAMPLE 8

(Synthesis of 6-(Phenylthio)uracil)

Reaction of 6-(phenylthio)-2,4-bis(benzyloxy)pyrimidine (400 mg, 1 mmol)with trimethylsilyl iodide (520 mg, 2.6 mmol) in CH₂Cl₂ (15 mL) asdescribed in Example 2, gave 6-phenylthiouracil (190 mg, 86%), m.p.266°-267° C.; ¹H NMR (DMSO-d₆) δ7.04-7.25 (m, 5H, SPh), 7.86 (s, 1H,6-H), 11.32 and 11.41 (2 s, 2H, 2 NH, D₂O exchangeable). Anal.(C₁₀HN₂O₂S) C, H, N.

EXAMPLE 8

(Synthesis of 1-[(2-Hydroxyethoxy)methyl]-5-(phenylselenenyl)uracil(PSAU)

Phenylselenenyl chloride (1.14 g, 6 mmol) was dissolved in dry pyridine(15 mL) and then the 1-[(2-hydroxyethoxy)methyl]uracil (1.0 g, 5.37mmol) was added. The reaction mixture was stirred at 60° C. for 24 h.The mixture was allowed to cool to room temperature and thenconcentrated in vacuo to remove pyridine. The residue was coevaporatedwith benzene (2×10 mL) and then with absolute EtOH (10 mL). The residuewas loaded onto a silica gel column and eluted first with CHCl₃ toremove residual diphenyl diselenide. The product was then obtained byelution with CHCl₃:MeOH (95:5) and the TLC pure fractions were pooledand concentrated. The solid residue was recrystallized from absoluteEtOH to yield the desired product as a white crystalline solid (1.4 g,76%); m.p. 118°-120° C.; ¹H NMR (DMSO-d₆) δ1.60 (s, 1H, OH, D₂Oexchangeable), 3.68-3.74 (m, 4H, OCH₂CH₂O), 5.17 (s, 2H, NCH₂O),7.26-7.57 (m, 6H, SePh and C-6 H), 8.37 (s, 1H, NH, D₂O exchangeable).Anal. (Cl₃H₁₄N₂O₄Se) C, H, N.

EXAMPLE 9

(Synthesis of 1-(Ethoxymethyl)-5-(phenylselenenyl)uracil)

Reaction of phenylselenenyl chloride (1.15 g, 6 mmol) with1-(ethoxymethyl)uracil (850 mg, 5 mmol) in pyridine (25 mL) as describedabove in Example 8 yielded the title compound (1.20 g, 74%); m.p.143°-145° C.; ¹H NMR (CDCl₃) δ1.20 (t, 3H, CH₃CH₂O), 3.67 (q, 2H,CH₃CH₂O), 5.08 (s, 2H, NCH₂O), 7.22-7.53 (m, 5H, SePh and C-6 H), 8.94(s, 1H, NH, D₂O exchangeable). Anal. (C₁₃H₁₄N₂O₃Se) C, H, N.

The novel enzyme inhibiting compounds of the invention and other usefulenzyme inhibiting compounds were evaluated for their ability to inhibitDHUDase and UrdPase. Further, toxicity of these compounds to host tissuewas also assessed. The testing conducted and the data obtained arediscussed and presented in the examples and tables that follow.

EXAMPLE 10

Mouse livers were obtained from female Swiss Albino (CD1) mice weighing20-24 g (Charles River Laboratories, Boston, Mass.). Mice weresacrificed by cervical dislocation and the livers removed, weighed,minced, and homogenized in ice-cold (3:1, v/w) buffer [20 mM potassiumphosphate. pH 8.0; 1 mM dithiothreitol (DTT), 1 mM EDTA] using aPolytron homogenizer (Brinkman Instruments, Westbury, N.J.). Thehomogenates were centrifuged at 105,000×g for 1 h. at 4° C. Thesupernatant fluids (cytosol) were collected and used as an enzymesource.

All assays described below were conducted at 37° C. under conditionswhere enzyme activity was linear with respect to time and enzymeconcentration. For each inhibitor, 5 concentrations were used rangingfrom 8-900 μM. Reactions were started by the addition of extract andstopped by boiling in a water bath for 2 minutes followed by freezing,Precipitated proteins were removed by centrifugation. Substrates wereseparated from products in the supematant by TLC and the radioactivityin the spots was determined on a percentage basis using a BertholdLB-2821 Automatic TLC-Linear Analyzer.

Pyrimidine nucleoside phosphorylases (dThdPase and UrdPase)

Nucleoside cleavage was measured isotopically by following the formationof nucleobases from their respective nucleosides as previouslydescribed. The reaction mixture contained 20 mM potassium phosphate (pH8), 1 mM EDTA, 1 mM DTT, 1 mM [2-¹⁴C]thymidine (56 Ci/mol) and 25 μLcytosol in a final volume of 50 μL. The incubation was terminated after30 min. Uridine and thymidine were separated from their respectivenucleobases on silica gel TLC plates developed with CHCl₃:MEOH:AcOH(90:5:5, v/v/v). The R_(f) values were uridine, 0.07; uracil, 0.43;thymidine, 0.14; and thymine, 0.62.

DHUDase

The activity of the enzyme was measured by following the formation ofdihydrouracil, carbamyl-β-alanine, and β-alanine from [6 -¹⁴C]uracil aspreviously described. The reaction mixture contained 20 mM potassiumphosphate (pH 8), 1.0 mM EDTA, 2 mM DTT, 5 mM MgCl₂, 25 μM [6-¹⁴C]uracil(56 Ci/mol), 100 μM NADPH and 25 μL of cytosol in a final volume of 50μL. The incubation was terminated after 15 min. Uracil, dihydrouracil,carbamyl-β-alanine, and β-alanine were separated on cellulose TLC platesdeveloped in the top phase of a mixture of nBuOH:H₂O:ammonia (90:45:15,v/v/v). R_(f) values were dihydrouracil 0.46; uracil 0.23; β-alanine andcarbamyl-β-alanine 0.09. DHUDase activity was determined as the sum ofthe products dihydrouracil, carbamyl-β-alanine, and β-alanine.

Kinetic studies

Determination and significance of apparent K_(i) values was pertbrinedusing uridine (1 mM) and 5 different concentrations of the inhibitorranging from 50-900M. Apparent K_(i) values were estimated from Dixon'splots (1/v vs. [I]) of the data by a computer program with least squaresfitting. Apparent K_(i), values are related to K_(i) values by thefollowing equation:

Apparent K_(i)=K_(is)(1+[S]/K_(m)/)1+([S]/K_(m)) (K_(is)/K_(ii))

where K_(is) and K_(ii) are inhibition constants that would have beenestimated from the replot of slope and intercept, respectively, of aLineweaver-Burk plots vs. [I]. If a compound is a competitive inhibitorwith respect to uridine, K_(ii)=∝ and K_(is)=K_(i). Therefore, theapparent .K_(i)=K_(i)(1+[S]1 K_(m)). Thus, tbr UrdPase from mouse liverwhich has a K_(m) value of 66 μM for uridine, the apparent K_(i), of acompetitive inhibitor, measured at uridine concentration of 1 mM, isapproximately 16-fold higher than their respective K_(i) values. Itshould be noted, however, that we have not characterized the compoundsused in this study with regard to the type of inhibition (competitive,noncompetitive, or uncompetitive) or whether they are substrates for theenzyme.

Protein concentrations were determined spectrophotometrically by themethod of Bradford using bovine γ-globulin as a standard.

Data obtained are illustrated below in Tables 1 and 2.

TARLE 1 Apparent inhibition constants of different compounds withenzymes isolated from mouse liver. Enzyme (Apparent K_(i),μM ± S.D.)Inhibitor UrdPase DHUDase 5-Phenylselenenyluracil (PSU) 205 ± 35  4.8 ±0.6 5-(Phenylselenenyl)uridine 4.0 ± 0.2 —5-(Phenylselenenyl)-2′-deoxyuridine 5.5 ± 0.6 — PSAU 3.8 ± 0.8 *1-Ethoxymethyl-5- 313 ± 32  * phenylselenenyluracil 5-Phenylthiouracil(PTU) 744 ± 85  5.4 ± 0.6 6-(Phenylselenenyl)acyclouridine 19.3 ± 1.5  —6-(Phenylselenenyl)acyclo-5-FUrd 35.0 ± 5.6  — 5-Benzylacyclouridine(BAU)  3.1 ± 0.22 —

TABLE 2 Inhibition constants (K_(is) ) of PSAU on hepatic uridinephosphorylase from different species Inhihibitor Mouse Liver Human LiverMonkey Liver BAU 420 ± 40 1190 ± 200 333 ± 49 PSAU 163 ± 9  340 ± 19 128± 14 K_(is) (in nM) ± standard error of estimation measured at 20 mMinorganic phosphate, 30-700 μM uridine and inhibitor concentrationsranging from 50-900 nM.

EXAMPLE 11

Pharmacokinetics of 1-[(2-hydroxyethoxy)methyl]-5-(phenylselenyl)uracil(PSAU) in CD-1 mice

PSAU was injected i.p. into female CD-1 mice. At various time intervals,250 μl of whole blood were collected from the orbital sinuses from threemice by a heparinized Natelson pipet and placed on ice. The whole bloodwas then centrifuged for 5 minutes to separate the plasma which was keptin a −20° C. freezer until preparation for analysis by the HPLC.

The pharmacokinetic parameters were estimated by compartmentalmodel-independent methods using a SIPHAR/Base program. The AUC wasdetermined by the trapezoidal rule with extrapolation to time infinityusing the terminal disposition slope (K) generated by a weightednonlinear least-squares regression of an exponential fit of the data,with the weighted square factor set as the reciprocal of the calculatedconcentration squared. Elimination half-life of uridine was calculatedfrom 0.693/K The total plasma clearance (C1) was calculated by dividingthe dose by the AUC and the weight of the mouse. The peak plasmaconcentration (C_(max)) values and time to peak plasma concentration(T_(max)) values were observed experimental values. Renal clearance(CL_(R)) of uridine was calculated by dividing the dose by the AUC. Thedata obtained are illustrated below in Table 3.

TABLE 3 Pharmacokinetics of PSAU in CD-1 Mice Dose of PSAU C_(max) AUCCL Apt½ ApV_(d) MRT 30 mg/kg 100.0 96.91 0.310 0.7 0.331 1.282 60 mg/kg210.0 349.00 0.173 1.4 0.356 2.275 C_(max) is peak plasma concentration[μM]; AUC is area under the curve (μmol × hr/ml); CL is total plasmaclearance (ml/hr/kg); Apt½ is elimination half-life (hr); V_(d) isvolume of distribution (L/kg); MRT is mean residence time (hr).

EXAMPLE 12

Effect of PSAU on the Pharmacokinetics of Plasma Uridine in CD-1 mice

PSAU was injected i.p. into female CD-1 mice. At various time intervals,250 μl of whole blood were collected from the orbital sinuses from threemice by a heparinized Natelson pipet and placed on ice. The whole bloodwas then centrifuged for 5 minutes to separate the plasma which was keptin a −20° C. freezer until preparation for analysis by the HPLC. Thepharmacokinetics were analyzed by the procedures noted in Example 11,and the data obtained are presented in Table 4.

TABLE 4 Nor- Dose of mal PSAU Conc. C_(max) T_(max) AUC CL Apt½ ApV_(d)MRT 30 mg/kg 2.58  8.1 1.2  74.95 0.408 5.3 3.032  8.045 60 mg/kg 3.0714.6 2.5 113.12 0.601 7.2 4.029 11.068

Materials

Melting points were determined on an Electrothermal IA 8100 digitalmelting point apparatus and are uncorrected. ¹H NMR spectra wererecorded on a General Electric QE-300 (300 MHz) spectrometer.Experiments were monitored using TLC analysis performed on Kodakchromatogram sheets precoated with silica gel and a fluorescentindicator, while column chromatography, employing silica gel (60-200mesh; Fisher Scientific, Fair Lawn, N.J.) was used for the purificationof products. Tetrahydrofuran (THF) was freshly distilled from the sodiumbenzophenone salt. LDA (2.0M), n-BuLi (1.6M), diphenyl diselenide,diphenyl disulfide, and trimethylsilyl iodide and other chemicals werepurchased from Aldrich Chemical Company (Milwaukee, Wis.). Microanalyseswere performed at Atlantic Microlabs (Atlanta, Ga.). [2-¹⁴C]Uridine (56Ci/mol), [2-¹⁴C]thymidine (56 Ci/mol), and [6-¹⁴C]uracil (56 Ci/mol)were obtained from Moravek Biochemicals Inc., Brea, Calif.;[6-¹⁴C]orotate (46.9 Ci/mol) from New England Nuclear Research Products,DuPont Co., Boston, Mass.; silica gel G/W₂₅₄ polygram, polyethyleneiminecellulose 300 PEI/UV₂₅₄ and cellulose CEL 300 UV polygram thin layerchromatography plates from Brinkman, Westbury, N.J.; protein assay kitfrom Bio-Rad Laboratories, Richmond, Calif. All other chemicals wereobtained from Sigma Co., St. Louis, Mo.

It should be clear that various modifications, additions andsubtractions can be made without departing from the spirit or scope ofthe invention. For example, it should be appreciated that the presentinvention can also be employed in conjunction with otherchemotherapeutic agents or biological response-modifying agents. Forexample, the combination therapy of the present inventions can beemployed in tandem with the administration of bone marrow stimulatingfactors, such as granulocyte-macrophage colony stimulating factors(GM-CFSs), other colony stimulating factors, erythropoietin (EPO) andother compounds that stimulate hematopoietic activity. (For a furtherdiscussion of GM-CSF activity, see Hammer et al, Antimicrobial Agentsand Chemotherapy, 31:1046-1050 (1987). Similarly, the combinationtherapy of the present invention can be undertaken in conjunction withefforts to stimulate the immune system, such as by the administration ofinterferons (e.g., alpha-A interferon) or other lymphokines.

All references cited above are expressly incorporated by referenceherein.

What is claimed is:
 1. A compound represented by the formula

where X is S or Se; Y is H, I, F, Cl, Br, methoxy, benzyl,selenenylphenyl, or thiophenyl, and R₁ is H or an acyclo tail having thegeneral formula

where R₂ is H, CH₂OH, or CH₂NH₂; R₃ is OH, NH₂ or OCOCH₂CH₂CO₂H; and R₄is O, S, or CH₂, provided when Y is H, then R₁ is an acyclo tail asabove or when R ₁ is H, then Y is I, F, Cl, Br, methoxy, or benzyl. 2.5-(phenylselenenyl)uracil (PSU).
 3. The compound of claim 1 which is1-[(2-hydroxyethoxy)methyl]-5-(phenylselenenyl)uracil (PSAU).
 4. Thecompound of claim 1 which is5-(phenylselenenyl)-2,4-bis(benzyloxy)pyrimidine.
 5. The compound ofclaim 1 which is 1-(ethoxymethyl)-5-(phenylselenenyl)uracil. 6.5-(phenylthio)uracil (PTU).
 7. The compound of claim 1 which is1-[(2-hydroxyethoxy)methyl]-5-(phenylthio)uracil. (PTAU).
 8. Thecompound of claim 1 which is5-(phenylthio)-2,4-bis(benzyloxy)pyrimidine. 9.5-(phenylselenenyl)barbituric acid.
 10. The compound of claim 1 17whichis 1-[2-(hydroxy-ethoxy)methyl]-5-(phenylselenenyl)barbituric acid. 11.The compound of claim 1 which is5-(phenylselenenyl)-2,4-bis(benzyloxy)barbituric acid.
 12. The compoundof claim 1 which is 1-(ethoxymethyl)-5-(phenylselenenyl)barbituric acid.13. 5-(phenylthio)barbituric acid.
 14. The compound of claim 1 17whichis 1-[(2-hydroxy-ethoxy)methyl]-5-(phenylthio)barbituric acid.
 15. Thecompound of claim 1 which is 5-(phenylthio)-2,4-bis(benzyloxy)barbituricacid.
 16. A pharmaceutical composition comprising a compound representedby the formula

where X is S or Se; Y is H, I, F, Cl, Br, methoxy, benzyl,selenenylphenyl, or thiophenyl, and R₁ is H or an a cyclo acyclo tailhaving the general formula

where R₂ is H, CH₂OH, or CH₂NH₂; R₅ is OH, NH₂ or OCOCH₂CH₂CO₂H; and R₄is O, S, or CH₂, provided when Y is H, then R₁ is an acyclo tail asabove or when R ₁ is H, then Y is I, F, Cl, Br, methoxy, or benzyl and apharmaceutically acceptable carrier.
 17. A compound represented by theformula

where X is S or Se; Y is H, I, F, Cl, Br, methoxy, benzyl,selenenylphenyl, or thiophenyl, and R ₁ is H or an acyclo tail havingthe formula

where R ₂ is H, CH ₂ OH, or CH ₂ NH ₂ ; R ₃ is OH, NH ₂ or OCOCH ₂ CO ₂H; and R ₄ is O, S, or CH ₂ , provided when Y is H, then R ₁ is anacyclo tail as above or when R ₁ is H, then Y is I, F, Cl, Br, methoxy,benzyl, selenenylphenyl, or thiophenyl.
 18. A pharmaceutical compositioncomprising a compound represented by the formula

where X is S or Se; Y is H, I, F, Cl, Br, methoxy, benzyl,selenenylphenyl, or thiophenyl, and R ₁ is H or an acyclo tail havingthe formula

where R ₂ is H, CH ₂ OH, or CH ₂ NH ₂ ; R ₃ is OH, NH ₂ or OCOCH ₂ CO ₂H; and R ₄ is O, S, or CH ₂ , provided when Y is H, then R ₁ is anacyclo tail as above or when R ₁ is H, then Y is I, F, Cl, Br, methoxy,benzyl, selenenylphenyl, or thiophenyl and a pharmaceutically acceptablecarrier.